Escherichia coli (“E. coli”) is a commonly used host for expression of proteins for research, diagnostic, therapeutic, and industrial purposes. Modern expression systems are capable of achieving high levels of a wide variety of proteins (Baneyx, Current Opinion in Biotechnology 10:411-421 (1999)). However, the quality of the expressed protein is often as important or more important than quantity. Proteins expressed in E. coli may be formed as insoluble aggregates, or they may have misincorporated amino acids (Bogosian, et al., Journal of Biological Chemistry 264:531-539 (1989)) or retain the N-terminal methionine (Chaudhuri, et al., Journal of Molecular Biology 285:1179-1194 (1999); Vassileva-Atanassova, et al, Journal of Biotechnology 69:63-67 (1999); Yamashita, et al., Protein Expression & Purification 16:47-52 (1999)). In addition, undesired post-translational modifications may occur such as oxidation (Berti, et al., Protein Expression & Purification 11:111-118 (1997); Konz, et al., Biotechnology Progress 14:393-409 (1998)) or α-N-6-phosphogluconoylation (Geoghegan, et al., Anal. Biochem. 267:169-184 (1999); Kim et al., Acta Crystallographica Section D-Biological Crystallography 57:759-762 (2001); Yan, et al. Biochemical & Biophysical Research Communications 262:793-800 (1999) “Yan et al. I;” Yan, et al., Biochemical & Biophysical Research Communications 259:271-282 (1999) “Yan et al. II”). These modifications may adversely effect activity, stability, structure, or immunogenicity of the expressed protein, greatly reducing the utility of E. coli as a host for polypeptide expression.
Alpha(α)-N-6-phosphogluconoylation of several recombinant proteins fused to hexahistidine affinity tags (“hexa His-tag”) has been described (Geoghegan, et al; Kim, et al.; Yan et al. I; Yan et al. II). In these studies, a gluconic acid derivative was found to attach to the end terminus of the recombinant protein. All of these proteins were expressed in B strains of E. coli using pET-based vectors (Novagen). Where reported, LB medium was used. The adduct was detected as an extra mass associated with the polypeptide of either 258 Daltons (“Da”), representing the addition of 6-phosphogluconolactone (6-PGL), or 178 Da, representing the presence of gluconolactone without the phosphate. Phosphogluconoylation was presumed to occur at the N-terminal α-amino group through reaction with endogenous 6-PGL, an intermediate of the pentose phosphate shunt. The +178 Da adduct was proposed to be the result of enzymatic activity acting on the +258 Da adduct to remove the phosphate. Formation of the adduct was shown to be specific to the amino acid sequence at the N-terminus adjacent to the His-tag. Polypeptide sequences of GXXHHHH, where XX is SS, SA, AS, or AA, were the most prone to α-N-6-phosphogluconoylation, whereas SHHHHHH was less prone, and PHHHHHH and PFHHHHHH were not modified at all (Geoghegan, et al.). Modifications at other amino groups elsewhere on the protein were not detected in vivo or in in vitro experiments that used high levels of added gluconolactone. (Geoghegan, et al.)
N-terminal phosphogluconoylation has been shown to inhibit crystallization of proteins (Kim, et al.), but relatively little else is known about its effect on protein function, stability, or immunogenicity. It is expected that 6-PGL, being a potent electrophile, may be involved in glycation reactions in vivo (Rakitzis and Papandreou, Chemico-Biological Interactions 113:205-216 (1998)). Glycation of proteins has been widely studied and is known to play a major role in aging and disease states related to diabetic complications (Baynes and Monnier, The Maillard Reaction in Aging, Diabetes, and Nutrition. Alan R. Liss, New York (1989)). Exogenously added delta-gluconolactone has been shown to cause glycation of hemoglobin, which may be a factor in the vascular complications of diabetes (Lindsay, et al., Clinica Chimica Acta 263:239-247 (1997)). Furthermore, glycation of alanine aminotransferase at the epsilon-amino group of Lys313 markedly reduces its catalytic activity (Beranek, et al., Molecular & Cellular Biochemistry 218:35-39 (2001)).
6-Phosphogluconolactonase (“pgl”) has been shown to be an essential enzyme of the pentose-phosphate pathway, specifically in the hydrolysis of 6-PGL to 6-phosphogluconic acid. (Miclet, et al., J. Biol. Chem. 276:34840-34846 (2001)) The gene encoding this enzyme has been identified in human (Collard, et al., FEBS Letters 459:223-226 (1999)), Pseudomonas aeruginosa (Hager, et al., Journal of Bacteriology 182:3934-3941 (2000)), and Trypanosoma brucei and Plasmodium falciparum (Miclet, et al.). Although pgl activity has long been observed in E. coli (Kupor and Fraenkel, Journal of Bacteriology 100:1296-1301 (1969) “Kupor I” and Kupor and Fraenkel, Journal of Biological Chemistry 247:1904-1910 (1972) “Kupor II”), no gene sequence responsible for encoding an enzyme with this activity has been identified (Cordwell, S. J. Arch. Microbiol. 172:269-279 (1999)). It has been suggested that in addition to enabling metabolic flux through the pentose phosphate shunt, pgl activity inside the cell prevents accumulation of 6-PGL and consequential damaging reactions with intracellular nucleophiles (Miclet, et al.). Reported observations of phosphogluconoylation of proteins at the N-terminus supports the hypothesis that the 6-PGL produced in the pathway can modify proteins, but there has been no reported evidence that modulating pgl activity can affect the levels of modified protein.
Furthermore, Escherichia coli strain BL21 (DE3) is a commonly used host for expression of proteins for research, diagnostic, therapeutic, and industrial purposes Studier, F. W., and Moffatt, B. A., J. Mol. Biol. 1986 May 5; 189(1):113-130. This strain is commercially attractive because it achieves very high expression levels of recombinant protein by means of coupling expression of a chromosomal copy of the T7 RNA polymerase and the use of a plasmid based T7 RNA polymerase promoter on the recombinant protein of interest. Accordingly, since the 17 RNA polymerase is an extremely selective and active RNA polymerase, transcription of the recombinant protein accumulate to very high levels, often even to the extent that host cell transcripts are diminished.
Unfortunately, the strain BL21 (DE3) releases very low, yet detectable, infectious lambda phage particles on the order of 10-20 plaque forming units/ml (PFU), Stewart Shuman, Proc. Natl. Acad. Sci. USA 1989; 89:3489-3493. Apparently, the T7 RNA polymerase gene was introduced into the BL21 host cell chromosome by transduction with the defective lambda phage DE3 carrying the T7 RNA polymerase gene inserted into the lambda int gene Studier, F. W., and Moffatt, B. A., J. Mol. Biol. 1986; 189(1):113-130. The resulting defective prophage cannot replicate normally due to the int gene interruption. Chromosomal excision of the DE3 prophage is a requirement for, the replication of the prophage prior to packaging and release of infectious phage particles and is dependant on homologous excisional recombination that is directed by the product of the int gene. As described herein very low levels of phage particles released are due to abnormal, int independent random prophage excision events. While the release of low levels of infectious phage particles may be acceptable in some research laboratories, any release of infectious phage is totally unacceptable in the biopharmaceuticals manufacturing plant setting both because of considerations for patient safety and because release of infectious agents can put at risk other E. coli based manufacturing processes.
A method for expressing or overexpressing polypeptides in a microorganism, such as E. coli, with a reduced incidence of phosphogluconoylation during fermentation is greatly needed. In addition, creation of a totally phage free BL21 (DE3) host cell would be highly desirable for the manufacture of therapeutic recombinant proteins in the E. coli format.